CN106699896B

CN106699896B - Tumor killing polypeptide capable of self-assembling into hydrogel and application thereof

Info

Publication number: CN106699896B
Application number: CN201611105009.4A
Authority: CN
Inventors: 邹枕玮; 金红林; 陈静; 伍钢; 杨坤禹; 胡建莉; 彭纲; 冯觉平; 李品东; 赵桂芳
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2016-12-05
Filing date: 2016-12-05
Publication date: 2020-06-05
Anticipated expiration: 2036-12-05
Also published as: CN106699896A

Abstract

The invention relates to a tumor killing polypeptide capable of self-assembling into hydrogel, which comprises a C-terminal self-assembly domain and an N-terminal killing domain, wherein the self-assembly domain and the killing domain are connected through a flexible domain, and the sequence of the self-assembly domain is (RADA)_7‑9. The tumor killing polypeptide capable of self-assembling into the hydrogel does not influence the function of self-assembling into the hydrogel, and has the capability of killing tumors. The sustained-release medicament can be directly implanted into a tumor body, a tumor side or a postoperative residual cavity through repeated local injection, open operation is not needed to expose the tumor, complications such as infection, bleeding and the like are reduced, and the physical and economic burden of a patient is reduced. The self-assembly polypeptide material can load chemotherapeutic drugs to directly kill tumors, and can also load tumor antigen protein, tumor lysate or tumor RNA to be used as tumor nano vaccines.

Description

Tumor killing polypeptide capable of self-assembling into hydrogel and application thereof

Technical Field

The invention relates to the field of anti-tumor, in particular to a tumor killing polypeptide capable of self-assembling into hydrogel.

Background

The main problems of the prior malignant tumor treatment are that ① tumor tissues are not clear with normal brain tissues, the operation total resection and precise radiotherapy in the true sense are difficult to carry out, the residual tumor tissues become the root of local recurrence, ② chemotherapy drugs need large dose, the tumor local drug concentration is low, the curative effect is poor, the systemic toxic and side effect is obvious, and patients cannot tolerate the treatment.

With the development of material science and biomedicine, the use of biomaterials as Drug carriers to load chemotherapeutic drugs such as paclitaxel, cisplatin and adriamycin and the like has made a promising progress in achieving local targeted Drug delivery of tumors to treat solid malignant tumors, which not only can enhance targeting and sustained release of drugs, but also can reduce systemic toxicity of chemotherapeutic drugs, Gliadel implant (Gliadel Wafer, u.s.a. Guilford company) is the first Drug-loaded degradable polymer for treating brain glioma worldwide, Gliadel Wafer has a diameter of 1.45cm and a thickness of about 1mm, and contains 192.3mg of polifeprosan20 (polifeprosan20) and 7.7mg of carmustine per tablet, u.a. Food and Drug Administration (Food and Drug Administration, FDA) approves the adaptation thereof as a new surgery and adjuvant radiotherapy adjuvant Drug for patients diagnosed with malignant glioma, and also can be used as surgery adjuvant Drug for patients with relapsed glioblastoma multiforme, u.a. the u.a. cancer center (FDA) has approved surgery and found that there is no clinical adjuvant Drug delivery, and no recurrence of gliadelphia Drug carrier, such as no Drug carrier, no Drug carrier is found in the clinical trial of gliadelphia tuberculosis, no recurrence, no Drug carrier is found in the clinical trial of Gliadel implant, no such as gladel 20.

Polypeptide hydrogels are gel materials formed by self-assembling polypeptide molecules triggered by specific conditions. Polypeptide molecules can spontaneously aggregate to form a Beta sheet structure after being dissolved in water, cations such as Ca2+, Mg2+, H + and the like can promote the polypeptide molecules to self-assemble to form nano fibers with a net structure, and the appearance of the nano fibers is gel. The material can be directly injected into a body to form gel, has good biocompatibility, the degradation product is amino acid, does not generate immune reaction and inflammatory reaction, and has no cytotoxicity. Currently, RADA-16 is one of the most commonly used self-assembled polypeptide materials, in which a functional peptide fragment (e.g., IKVAV) is attached to RADA-16 molecules by solid phase synthesis, and the gel material has the same biological activity as the functional peptide fragment after the polypeptide molecules are self-assembled, but has the following disadvantages: 1) no antitumor activity by itself; 2) RADA-16 can form a gel, but if the amino acid sequence of the carried functional peptide fragment is too long (such as melittin: GIGAVLKVLTTGLPALISWIKRKRQQ), functional peptide fragments would disrupt the spontaneous aggregation of RADA-16 to form the Beta sheet architecture and would not self-assemble into a gel.

With the rapid development of nanotechnology, optical tumor therapy, such as photothermal therapy, has been rapidly developed for application in tumor therapy. Photothermal therapy is to generate heat to kill tumor cells by irradiating a substance having light-absorbing ability with laser light. Indocyanine green (ICG) is an FDA-approved drug for clinical use, and also plays a significant role in photothermal therapy. However, the instability of ICG in aqueous solution and its rapid clearance in vivo limits its use.

Therefore, a drug-loaded material which is safe, good in biocompatibility, degradable and has anti-tumor activity is developed, and the drug-loaded material is combined with photo-thermal therapy for treating malignant tumors, so that the drug-loaded material has a wide clinical application prospect.

Disclosure of Invention

In order to solve the above problems, the present invention provides a tumor killing polypeptide capable of self-assembling into a hydrogel, comprising a C-terminal self-assembly domain and an N-terminal killing domain, wherein the self-assembly domain and the killing domain are connected by a flexible domain, and the sequence of the self-assembly domain is (RADA)_7-9。

When the RADA unit of the self-assembly domain is lengthened to 28 peptides (i.e., 7 repeats) to 36 peptides (i.e., 9 repeats), the resulting self-assembly domain can be unexpectedly linked to some cell-killing polypeptides, without affecting its self-assembly hydrogel function, and imparting its own tumor killing ability.

Preferably, the killing domain is one or more combination selected from KLA with sequence KLAKLAKKLAKLAK, melittin with sequence GIGAVLKVLTTGLPALISWIKRKRQQ.

Preferably, the flexible domain is GG.

The invention also discloses application of the tumor killing polypeptide capable of self-assembling into hydrogel in preparation of sustained-release medicaments.

The invention also discloses an anti-tumor hydrogel sustained-release medicament which is obtained by carrying anti-tumor medicaments on the tumor killing polypeptide capable of self-assembling into hydrogel.

Preferably, the anti-tumor drug is indocyanine green, doxorubicin, a chemotherapeutic drug and a tumor vaccine

The invention also discloses a preparation method of the anti-tumor hydrogel sustained-release medicament, which comprises the following steps:

s1: preparing an aqueous solution of the tumor killing polypeptide capable of self-assembling into hydrogel and an aqueous solution of the anti-tumor drug;

s2: mixing the aqueous solution of the tumor killing polypeptide capable of self-assembling into hydrogel with the aqueous solution of the anti-tumor drug to obtain a mixed solution;

s3: and placing the mixed solution at low temperature to form hydrogel, thus obtaining the anti-tumor hydrogel sustained-release medicament.

Preferably, the concentration of the tumor killing polypeptide capable of self-assembling into hydrogel in the mixed solution is 10-30 mg/mL.

Preferably, the mixed solution is left at 4 ℃ for 2 hours to form a hydrogel.

The invention also has the following advantages:

① after RADA-16 is improved, active peptide fragments KLA and melittin do not interfere with gel formation, and both the RADA-KLA and the RADA-melittin which are obtained by design can be self-assembled to form a gel material with anti-tumor activity;

② the sustained release preparation can be directly implanted into tumor body or postoperative residual cavity by repeated local injection, without open operation to expose tumor, thereby reducing complications such as infection and hemorrhage, and reducing body and economic burden of patients.

③ the slow release agent forms gel under the trigger of body fluid, can completely fill postoperative residual cavity, give full play to the anti-tumor effect of material and medicine, further reduce recurrence;

④ the degradation product is amino acid, and does not generate immune reaction and inflammatory reaction;

⑤ has good viscoelasticity, and compression modulus similar to that of brain and spinal cord tissue, and is especially suitable for central nervous system tumor;

⑥ the preparation method is simple and suitable for mass production;

⑦ can load and slowly release loaded active molecules (ICG or DOX), which is helpful for maintaining the stability of active molecules in vivo, and can continuously exert synergistic killing effect on tumor and reduce the damage to other organs by implanting local slow release active molecules;

⑧ function expansion, the self-assembly polypeptide material can load chemotherapy drug to directly kill tumor, and also can load tumor antigen protein, tumor lysate, small interfering RNA, tumor molecule target drug or tumor RNA as tumor nanometer vaccine.

Drawings

FIG. 1 is a schematic diagram of the structure of a tumor killing polypeptide that can self-assemble into a hydrogel;

FIG. 2 is an electron micrograph of KLA-RADA hydrogel and Melitin-RADA hydrogel;

FIG. 3 is a statistical plot of the release times of RADA-melittin hydrogel and melittin alone;

FIG. 4 is a graph of the in vitro tumor cell killing ability of KRI hydrogel and MRI hydrogel studied using PI and DAPI double staining. C6 cells are inoculated in KRI hydrogel, MRI hydrogel, RADA16 and PBS, after 24 hours of culture, the cells in RADA16 and PBS grow well, and a large amount of tumor cell apoptosis (red) can be seen in the KRI hydrogel and MRI hydrogel group;

FIG. 5 is a statistical chart of in vitro tumor cell killing ability of RADA-melittin hydrogel quantitatively studied by MTT experiment;

FIG. 6 is a graph of photothermal effect of an MRI hydrogel in vitro;

FIG. 7 is a photograph of fluorescence images of MRI gel loaded ICG implanted in mice;

FIG. 8 is a photograph of the fluorescence images of organs and tumors obtained after implantation of an ICG loaded MRI gel into a mouse;

FIG. 9 is a temperature profile during laser irradiation of tumors inoculated with RADA-KLA + ICG, RADA16, PBS on day 2;

FIG. 10 is a statistical plot of the volume of mouse tumor bodies treated with and without laser irradiation after random injection of RADA-KLA-ICG, RADA16, PBS;

FIG. 11 is a graph showing the weight statistics of tumor bodies after the above-mentioned treatment;

FIG. 12 photograph of HE staining of tumor tissue.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The inventors tested various hydrogel materials including the RADA16 peptide during the course of experiments and found that they were not suitable for carrying the killing polypeptide. The inventors tried to increase the number of RADA units. Unexpectedly, when the number of RADA units was increased to 7-9 (i.e., RADA28 peptide-RADA 36 peptide), a killing polypeptide could be attached to the N-terminus of the resulting peptide sequence and still form a hydrogel, and the hydrogel was capable of carrying an anti-tumor drug. In the following, we will prepare some embodiments of the invention by exemplifying the RADA32 peptide, the structure of which tumor killing polypeptides that can self-assemble into a hydrogel is shown in fig. 1.

Synthesis of RADA-KLA and RADA-melittin

Polypeptide molecules required in the experiment are synthesized by an automatic polypeptide synthesizer, purified by high performance liquid chromatography, and the purity and the sequence of the polypeptide molecules are detected by a mass spectrometer, an amino acid analyzer and a polypeptide analyzer. The sequence of RADA-KLA is AcN-RADARADARADARADARADARADARADARADAGGKLAKLAKKLAKLAK-NH₂(SEQID NO:1), the purity of the synthesized polypeptide is more than 98%, the molecular weight is 4986.64 Da, the sequence of the RADA-melittin is AcN-RADARADARADARADARADARADARADARADAGGGIGAVLKVLTTGLPALISWIKRKRQ-NH₂(SEQ ID NO:2) with a purity of greater than 98% and a molecular weight of 6310.11 Da.

2. Construction of RADA-KLA hydrogel and RADA-melittin hydrogel

10mg of sterile RADA-KLA and RADA-melittin powder are respectively dissolved in 500 microliters of sterile triple distilled water to obtain colorless transparent RADA-KLA polypeptide solution and RADA-melittin polypeptide solution, 500 microliters of 1.8% NaCl solution is respectively added into the solution, and the mixed solution is placed at 4 ℃ for 2 hours.

3. Construction of KLA-RADA-ICG (KRI), Melitin-RADA-ICG (MRI) and Melitin-RADA-DOX (MRD) hydrogels

20mg of sterile RADA-KLA powder is dissolved in 500 microliters of sterile triple distilled water to obtain a 2% mass-volume RADA-KLA or RADA-melittin solution, and a 1mg/ml ICG solution (dissolved in a 1.8% NaCl solution) is prepared. 2% RADA-KLA and RADA-Melittin solutions were mixed with ICG solution in equal volumes, respectively, to obtain 1% polypeptide concentration of KLA-RADA-ICG (KRI) or Melitin-RADA-ICG (MRI) complex, and the mixture was left at 4 ℃ for 2 hours to form a gel.

20mg of sterile Melitin-RADA powder is dissolved in 500 microliters of sterile triple distilled water to obtain a RADA-Melitin solution with the mass-volume ratio of 2%, and a DOX solution (dissolved in a 1.8% NaCl solution) with the mass-volume ratio of 20mg/ml is prepared. After 2% RADA-KLA or RADA-Melittin solution and ICG solution are mixed in equal volume, a 1% polypeptide concentration Melitin-RADA-DOX (MRD) complex (containing DOX at a concentration of 10mg/ml) is obtained, and the mixture is placed at 4 ℃ for 2 hours to form the Melitin-RADA-DOX (MRD) hydrogel.

Slow Release of RADA-melittin hydrogel

1mL of RADA-melittin hydrogel and 1mL of melittin solution are respectively put into a dialysis bag with the molecular weight cut-off of 25 kD. The dialysis bag was placed in a beaker containing 500mL of a solution (0.9% NaCl, pH 7.4) and stirred with a magnetic bar at normal temperature (200 rpm). Samples (2. mu.L) were taken from the bag at different time points over a 48-hour period and assayed for polypeptide concentration using the BCA method. During the 48-hour dialysis, the dialysate was changed 8 times in total for a total volume of 4L. As shown in FIG. 3, the RADA-melittin group had a slow release profile compared to the melittin group alone.

Cell killing Capacity of MRI hydrogel and KRI hydrogel

In the PI and DAPI fluorescence double staining experiment, different volumes of MRI hydrogel (or KRI hydrogel), RADA hydrogel and PBS solution are added in advance into a 24-well plate, and the gel is directly paved on the surface of the well plate. Subsequently, 2X 10 of the above-mentioned well plates were added⁴Cells were incubated for 24 hours. After removing the medium, the cells were washed twice with PBS, followed by staining in a well plate for 30 seconds with calcein (calcein-AM, 2. mu.L/mL) and propidium iodide (PI, 3. mu.L/mL), followed by washing the cells twice with PBS, and finally with fluorescenceCells were observed under microscope for fluorescence, red for apoptotic and necrotic tumor cells and green for normal cells. As seen in fig. 4, the PBS and RADA hydrogel treated groups alone were observed as red cells, while the MRI hydrogel group was almost entirely red spots, indicating that the MRI hydrogel group was able to completely kill C6 cells. While KRI hydrogel group can observe partial red and partial green cells, which proves that KRI hydrogel can also have stronger killing effect on C6 cells.

MTT assay quantitative determination of cell killing ability of MRI hydrogel: different volumes of MRI hydrogel, RADA hydrogel, PBS solution were pre-loaded into 96-well plates, and the gels were directly plated onto the surface of the plates. Subsequently, 5X 10 of the above-mentioned well plates were added to each of the plates³Cells were incubated for 24 hours. After removal of the medium, the cells were washed three times with PBS, and then the surviving cells were assayed by MTT. As shown in FIG. 5, RADA hydrogel has a partial promotion effect on cell proliferation, while the killing of the MRI hydrogel on C6 cells is gradually enhanced with the increase of the volume, and the cell killing rate reaches 98% when 50 μ L of the MRI hydrogel is added, thereby fully proving the strong killing capability of the MRI hydrogel on tumor cells.

Photothermal dioxin of MRI hydrogel

mu.L of the MRI hydrogel, RADA hydrogel and PBS solution were added to a 10cm diameter cell culture dish to form a circular drop of water having a diameter of about 1 cm. Using a near infrared laser at 808nm and at 2W/cm²The droplets were irradiated for 3min at the power of (1) and the temperature change was recorded every 30 seconds. As shown in FIG. 6, under the laser irradiation treatment, the RADA hydrogel and the PBS solution did not have obvious temperature changes, while the MRI hydrogel had a large temperature rise, and the temperature of the central region of the MRI hydrogel was close to 80 ℃, which proves that the MRI hydrogel has strong photothermal conversion capability.

Slow Release and protective Effect of MRI hydrogel on ICG in vivo

Establishing a nude mouse subcutaneous transplantation tumor: at a ratio of 1X 10 per mouse⁶The inoculation amount of each C6 cell is planted in the subcutaneous of a nude mouse until the tumor volume reaches about 100mm³At about 10 days, 50. mu.L of MRI hydrogel and RADA hydrogel were injected.

MRI hydrogel and ICG solution with the concentration of 0.5mg/ml are respectively injected into a subcutaneous tumor body of a nude mouse, a fluorescence signal of a tumor part is detected by using a small animal fluorescence imaging system 24 hours after injection, and the result shows that the degradation of an individual ICG group is fast and the fluorescence signal is very weak at 24 hours. Whereas the MRI hydrogel group was able to maintain a strong ICG fluorescence signal clearly and accumulated mainly in the tumor part (fig. 7 and 8). The experimental results prove that the MRI hydrogel can protect and slowly release the ICG, provide a foundation for the photothermal therapy of the ICG and reduce the distribution of the ICG in normal tissues.

Anticancer animal experiments with MRI and KRI hydrogels

Establishing a nude mouse subcutaneous transplantation tumor: at a ratio of 1X 10 per mouse⁶The inoculation amount of each C6 cell is planted in the subcutaneous of a nude mouse until the tumor volume reaches about 100mm³75 μ L of each of MRI, KRI, RADA, and PBS was injected (about 10 days) and laser irradiated controls were prepared. Laser irradiation was started on day 2 while recording the temperature change of the tumor portion during irradiation, and the tumor was observed by photographing on day 3. Tumor sizes were measured on

days

2, 4, 6, 8, and 10 after laser irradiation, respectively. Functional verification of MRI hydrogel and KRI hydrogel were performed separately in two independent animal experiments.

The laser irradiation significantly increased the intratumoral temperature of the KRI hydrogel group by up to 22 deg.C (FIG. 9), while the temperature rises of the other treatment groups were all within 10 deg.C. The KRI hydrogel group tumor portion started to scab on day 2 after laser irradiation, indicating that KRI hydrogel had a photothermal effect in vivo.

Tumor volume remained low for mice treated with MRI hydrogel in combination with laser irradiation (fig. 10), and only 1 mouse had a small tumor at day 10. The MRI hydrogel-icg (ri) hydrogel-laser irradiation treatment group had a tumor volume smaller than that of the IRADA-icg (ri) hydrogel-laser irradiation treatment group, and the tumors of both groups were significantly different in volume and weight (fig. 11). As can be seen from HE tissue staining (fig. 12), compared to the PBS-treated group, the MRI hydrogel-treated group alone significantly caused apoptosis and necrosis of tumor cells, and aggregation of small nuclear immune cells, suggesting that the MRI hydrogel can promote apoptosis and necrosis of tumor cells in vivo.

Anticancer animal experiments with Melitin-RADA-DOX hydrogel

In addition to loading ICG, RADA-Melitin hydrogel can also be loaded with a tumor chemotherapeutic, such as DOX. Establishment of C57BL/6 subcutaneous transplantable tumors: at a ratio of 5X 10 per mouse⁶The inoculation amount of mouse melanoma B16 cells was inoculated under C57BL/6 subcutaneous tissue, 50. mu.L of Melitin-RADA-DOX hydrogel, RADA-Melitin hydrogel, and PBS were injected when the tumors grew for about 10 days, and the tumors were removed and photographed on the 10 th day after the drug injection. Compared with the PBS and RADA-Melitin hydrogel group, the Melitin-RADA-DOX hydrogel group has the advantage that the tumor volume is obviously reduced, and the effect of synergistically promoting and inhibiting tumors of Melitin and DOX is fully proved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology

<120> tumor killing polypeptide capable of self-assembling into hydrogel and application thereof

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<170>PatentIn version 3.5

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<212>PRT

<213> Artificial sequence

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Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala

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Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala

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Gly Gly Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys

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<213> Artificial sequence

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Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala

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Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala Arg Ala Asp Ala

20 25 30

Gly Gly Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro

35 40 45

Ala Leu Ile Ser Trp Ile Lys Arg Lys Arg Gln

50 55

Claims

1. A tumor killing polypeptide capable of self-assembling into hydrogel, which comprises a C-terminal self-assembly domain and an N-terminal killing domain, wherein the self-assembly domain and the killing domain are connected through a flexible domain, and the sequence of the self-assembly domain is (RADA)_7-9The killing structural domain is KLA or melittin, the sequence of the KLA is KLAKLAKKLAKLAK, and the sequence of the melittin is GIGAVLKVLTTGLPALISWIKRKRQQ.

2. The tumor killing polypeptide of claim 1, wherein the flexible domain is GG.

3. Use of a tumor killing polypeptide of claim 1 or 2 that is self-assemblable into a hydrogel in the preparation of a sustained release medicament.

4. An anti-tumor hydrogel sustained-release agent, which is obtained by carrying an anti-tumor drug with the tumor-killing polypeptide capable of self-assembling into a hydrogel according to claim 1 or 2.

5. The anti-tumor hydrogel sustained-release preparation according to claim 4, wherein the anti-tumor drug is a chemotherapeutic drug, a small interfering RNA (small interfering RNA) or a tumor molecule targeted drug.

6. The method for preparing the anti-tumor hydrogel sustained-release agent according to claim 4 or 5, comprising the steps of:

s1: preparing an aqueous solution of the tumor killing polypeptide capable of self-assembling into hydrogel and an aqueous solution of the anti-tumor medicament;

7. The method according to claim 6, wherein the concentration of the tumor killing polypeptide capable of self-assembling into the hydrogel in the mixed solution is 10-30 mg/mL.

8. The production method according to claim 6 or 7, wherein the mixed solution is left at 4 ℃ for 2 hours to form a hydrogel.